Possible involvement of phosphorylation of occludin in tight junction formation.
Possible involvement of phosphorylation of occludin in tight junction formation.
复制标题
occludin的磷酸化可能参与紧密的连接形成。
DOI：
10.1083/jcb.137.6.1393
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发表时间：
1997-06-16
期刊：
影响因子：
--
通讯作者：
Tsukita S
中科院分区：
文献类型：
--
作者：
Sakakibara A;Furuse M;Saitou M;Ando-Akatsuka Y;Tsukita S
封闭蛋白是一种位于上皮细胞和内皮细胞紧密连接处的膜蛋白。融合培养MDCK Ⅰ细胞的Occludin在SDS-PAGE上显示多条（>10条）分子量在62 ~ 82 kD之间的条带，其中2 ~ 3条最低分子量的条带占优势。在这些条带中，较低的主要条带基本上用1%NP-40提取，而其他较高的MR条带在NP-40不溶性级分中选择性地回收。碱性磷酸酶治疗收敛这些乐队的occludin在NP-40可溶性和不溶性馏分到最低的M r带，磷酸氨基酸分析确定磷酸丝氨酸（和磷酸苏氨酸弱）在较高的M r带的occludin。这些发现表明，磷酸化导致occludin条带上移，并且高度磷酸化的occludin抵抗NP-40提取。当细胞在低Ca培养基中生长时，几乎所有闭合蛋白都是NP-40可溶的。从低到正常的钙介质切换增加NP-40-不溶性occludin的量在10分钟内，然后逐渐上移的带。这种不溶解和带移与免疫荧光显微镜检测到的紧密连接形成时间相关。此外，我们发现，抗鸡occludin单克隆抗体，OC-3，不承认主要的低M r带的occludin（非或较少磷酸化形式），但特异性较高的M r带（磷酸化形式）的免疫印迹。免疫荧光显微镜显示，该单克隆抗体主要染色的紧密连接适当的肠上皮细胞，而其他抗occludin单克隆抗体，它可以识别占主导地位的较低的M r带，标记其基底外侧膜（和细胞质）以及紧密连接。因此，我们得出结论，非或较少磷酸化occludin分布在基底外侧膜和高度磷酸化occludin选择性地集中在紧juctions作为NP-40不溶性的形式。这些发现表明，闭合蛋白的磷酸化是紧密连接组装的关键步骤。
Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest M r were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher M r bands were selectively recovered in the NP-40–insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40–soluble and -insoluble fractions into the lowest M r band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher M r bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40–insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti–chicken occludin mAb, Oc-3, did not recognize the predominant lower M r bands of occludin (non- or less phosphorylated form) but was specific to the higher M r bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower M r bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40–insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.